PROJECT SUMMARY Zika virus (ZIKV), like dengue and West Nile viruses (DENV, WNV), is a flavivirus, but among these, only ZIKV regularly causes microcephaly and other birth defects, despite the fact that WNV, for example, is also highly neurotrophic. The reason for this unique pathology of ZIKV is unknown. This application proposes to study the mechanisms by which ZIKV, but not other flaviviruses, infects placental barrier cells, and validate our results in vivo in a murine pregnancy model. To access fetal tissues, a virus must be transported into the fetal circulation, which necessitates crossing of a placental barrier composed of syncytiotrophoblasts, cytotrophoblasts and fetal endothelial cells. Of these, only fetal endothelial cells are in direct contact with the fetal bloodstream. Our preliminary studies make four key points. First, ZIKV infects fetal endothelial cells with dramatically higher efficiency than does DENV or WNV. Second, the receptor tyrosine kinase AXL is the predominant flavivirus entry cofactor present on these cells. Third, ZIKV utilizes AXL much more efficiently than does DENV or WNV. Fourth, AXL promotes ZIKV infection at attachment and post-attachment steps in the viral lifecycle. Together, these observations suggest the hypothesis that ZIKV crosses the placental barrier by infecting fetal endothelial cells in an AXL- dependent manner. Therefore, in the studies proposed here, we will define the molecular determinants of the differential use of AXL by ZIKV and WNV (Aim 1). We will also investigate the post-attachment events resulted from the AXL engagement by ZIKV (Aim 2). Finally, we will seek to demonstrate in a mouse pregnancy model that efficient use of AXL allows ZIKV to breach the placental barrier, whereas WNV is prevented from entering fetal circulation because, due to its inefficient use of AXL, it cannot infect fetal endothelial cells (Aim 3). Collectively, the results of these studies will provide important insights into the properties of ZIKV that allow it to access the fetal circulation and cause microcephaly.